Background Circulating tumor cells (CTCs) are liquid biopsies that represent micrometastatic disease and may offer unique insights into future recurrences in non-small cell lung cancer (NSCLC). Due to CTC rarity and limited stability, no stable CTC-derived xenograft (CDX) models have ever been generated from non-metastatic NSCLC patients directly. Alternative strategies are needed to molecularly characterize CTCs and means of potential future metastases in this potentially curable patient group. Methods Surgically resected NSCLC primary tumor tissues from non-metastatic patients were implanted subcutaneously in immunodeficient mice to establish primary tumor patient-derived xenograft (ptPDX) models. CTCs were isolated as liquid biopsies from the blood of ptPDX mice and re-implanted subcutaneously into naïve immunodeficient mice to generate liquid biopsy CTC-derived xenograft (CDX) tumor models. Single cell RNA sequencing was performed and validated in an external dataset of non-xenografted human NSCLC primary tumor and metastases tissues. Drug response testing in CDX models was performed with standard of care chemotherapy (carboplatin/paclitaxel). Blockade of MYC, which has a known role in drug resistance, was performed with a MYC/MAX dimerization inhibitor (10058-F4). Results Out of ten ptPDX, two (20%) stable liquid biopsy CDX mouse models were generated. Single cell RNA sequencing analysis revealed an additional regenerative alveolar epithelial type II (AT2)-like cell population in CDX tumors that was also identified in non-xenografted NSCLC patients’ metastases tissues. Drug testing using these CDX models revealed different treatment responses to carboplatin/paclitaxel. MYC target genes and c-MYC protein were upregulated in the chemoresistant CDX model, while MYC/MAX dimerization blocking could overcome chemoresistance to carboplatin/paclitaxel. Conclusions To overcome the lack of liquid biopsy CDX models from non-metastatic NSCLC patients, CDX models can be generated with CTCs from ptPDX models that were originally established from patients’ primary tumors. Single cell analyses can identify distinct drug responses and cell heterogeneities in CDX tumors that can be validated in NSCLC metastases tissues. CDX models deserve further development and study to discover personalized strategies against micrometastases in non-metastatic NSCLC patients.

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Background: Circulating tumor cells (CTCs) represent micrometastatic disease and may offer unique insights into future recurrences in lethal malignancies, including non-small cell lung cancer (NSCLC). Due to CTC rarity and limited stability, no CTC-derived xenograft (CDX) models have ever been generated from non-metastatic NSCLC patients directly. Alternative strategies are needed to molecularly characterize CTCs in this potentially curable patient group. Methods: Surgically resected NSCLC primary tumor tissues were implanted in immunodeficient mice to establish ten patient-derived xenografts (PDXs). CTCs from 2/10 PDX models led to generation of two stable metastatic models that were studied by single cell sequencing. Results: Single cell analysis revealed an additional alveolar epithelial type II (AT2) population in metastatic tumors, besides a common AT2 cluster in PDX/metastatic tumors. This was consistent with an external validation set analysis in primary and metastatic NSCLC patient tumors. Further, AT2 clusters of metastatic tumors expressed higher cancer stemness genes versus primary PDX tumor that was recapitulated in patients primary and metastatic tumors. Conclusions: Stable metastatic models from early stage NSCLC patients can be generated with CTCs from PDX models. The distinct AT2 population identified in CDX tumors with cancer stemness features might be critical mediator of metastasis that deserves further study to discover personalized strategies against NSCLC micrometastases. Citation Format: Kanve Nagaraj Suvilesh, Yulia I. Nussbaum, Vijay Radhakrishnan, Yariswamy Manjunath, Diego M. Avella, Kevin F. Staveley-O’Carroll, Eric T. Kimchi, Aadel A. Chaudhuri, Chi-Ren Shyu, Guangfu Li, Klaus Pantel, Wesley C. Warren, Jonathan B. Mitchem, Jussuf T. Kaifi. Xenograft models of non-metastatic non-small cell lung cancer reveals micrometastasis-associated single cell composition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 267.

Colorectal cancer (CRC) remains one of the deadliest malignancies worldwide despite recent progress in treatment strategies. Though immune checkpoint inhibition has proven effective for a number of other tumors, it offers benefits in only a small group of CRC patients with high microsatellite instability. In general, heterogenous cell groups in the tumor microenvironment are considered as the major barrier for unveiling the causes of low immune response. Therefore, deconvolution of cellular components in highly heterogeneous microenvironments is crucial for understanding the immune contexture of cancer. In this review, we assimilate current knowledge and recent studies examining anti-tumor immunity in CRC. We also discuss the utilization of novel immune contexture assessment methods that have not been used in CRC research to date.

Cytotoxic T lymphocyte (CTL) infiltration is associated with survival, recurrence, and therapeutic response in colorectal cancer (CRC). Immune checkpoint inhibitor (ICI) therapy, which requires CTLs for response, does not work for most CRC patients. Therefore, it is critical to improve our understanding of immune resistance in this disease. We utilized 2391 CRC patients and 7 omics datasets, integrating clinical and genomic data to determine how DNA methylation may impact survival and CTL function in CRC. Using comprehensive molecular subtype (CMS) 1 patients as reference, we found TBX21 to be the only gene with altered expression and methylation that was associated with CTL infiltration. We found that CMS1 patients with high TBX21 expression and low methylation had a significant survival advantage. To confirm the role of Tbx21 in CTL function, we utilized scRNAseq data, demonstrating the association of TBX21 with markers of enhanced CTL function. Further analysis using pathway enrichment found that the genes TBX21, MX1, and SP140 had altered expression and methylation, suggesting that the TP53/P53 pathway may modify TBX21 methylation to upregulate TBX21 expression. Together, this suggests that targeting epigenetic modification more specifically for therapy and patient stratification may provide improved outcomes in CRC.

146 Background: Colorectal cancer (CRC) is one of the deadliest malignancies worldwide. Though immune checkpoint inhibition has proven effective for a number of other tumors, it offers benefits in only a small group of CRC. In general, heterogenous cell groups in the tumor microenvironment (TME) are considered as the major barrier for unveiling the causes of low immune response. Therefore, deconvolution of cellular components in highly heterogeneous microenvironments is crucial for understanding those mechanisms. Single cell sequencing technology revolutionized TME research enabling profiling cells in high resolution. Methods: We have analyzed scRNA-seq data from 23 CRC patients with pre-treatment primary tumors using Seurat V3 pipeline. To investigate intercellular ligand-receptor interactions, we used CellPhoneDB and CellChat methods. The results of two independent analyses showed 4 CRC samples with no SPP1-CD44 interaction. It is known, that OPN which is the protein encoded by SPP1 gene, binds to CD44 and can cause cell survival, proliferation, and angiogenesis. Interestingly, analysis of the cellular composition of all 23 samples did not reveal differences in SPP1+ macrophages’ content for those 4 “no SPP1-CD44” samples. To investigate the mechanisms that could cause differences in SPP1-CD44 expression across the samples, we analyzed developmental trajectories of single cells using Slingshot trajectory inference method. Results: Ligand-receptor interactions analysis revealed 4 CRC samples that lacked SPP1-CD44 interaction that is known to be responsible for tumor progression in CRC. But the proportion of SPP1+ cells was not significantly different in those 4 samples compared to other samples. Trajectory inference analysis showed that the cells from “no SPP1-CD44” samples had high expression of anti-inflammatory macrophage markers in the end of the trajectory. While cells from “high SPP1-CD44” samples had high expression of pro-inflammatory macrophage markers at the same point. Conclusions: Based on our data-driven study, we suggest that SPP1+ macrophages’ heterogeneity may affect SPP1-CD44 interaction. Thus, targeting SPP1+ macrophages that have anti-inflammatory phenotype can potentially interrupt SPP1-CD44 interaction and therefore reduce tumor progression and immune suppression.

Background: Non-metastatic NSCLC treated with curative surgery has a five-year survival of ~50%, mostly due to development of recurrences. Despite being considered as potential drug screening platforms, patient-derived xenograft (PDX) models are inefficient due to low tumor engraftment rates and complex animal care. Patient-derived organoid (PDO) models overcome these limitations as potential clinically applicable drug testing platforms. Our objective was to develop PDO models from non-metastatic NSCLC patients to study epithelial cell type heterogeneity and drug sensitivities for precision medicine. Hypothesis: PDO models reliably capitulate the patient primary tumor and serve as versatile platforms for high-throughput screening of standard-of-care and repurposed drugs. Methods: Single cell suspensions prepared from resected lung tumor tissues from ten NSCLC patients (both adenocarcinoma and squamous cell carcinoma) were mixed with Matrigel (growth factor-reduced) and cultured in organoid growth medium. Organoids and matched primary tumors were compared by histopathology [H&E staining and immunohistochemistry for cytokeratin (CK) 5/6, CK7, Napsin A, Thyroid transcription factor-1 (TTF-1) and p40] and by bulk RNA sequencing. Upon passaging, PDOs were seeded in triplicates, treated with carboplatin/paclitaxel doublet chemotherapy, and drug responses were determined using bright-field 3D imaging (z-stack method). Tumor growth (%) was determined on day 3 and 6 of treatments. RNA sequencing analyses identified a potential drug targets and repurposed drug (aldoketoreductase inhibitor Epalrestat) was tested to overcome chemoresistance in PDOs. Results: NSCLC PDO growth was established from 11/12 (91.7%) primary tumors with a median time of 11 days (range 4-18 days) to reach volume of 100 μm3. PDOs retained histopathological features and biomarker expression of the matched tumors. As determined by growth differences (p<0.05), 5/9 (55.6%) PDOs were chemosensitive and 4/9 (44.4%) PDOs were chemoresistant against carboplatin/paclitaxel. Whole transcriptome analysis confirmed conservation of the epithelial cell composition in all PDOs in comparison to the matched primary tumors. Potentially druggable genes associated with chemotherapy resistance were identified and the aldoketoreductase inhibitor Epalrestat was repurposed and shown to be effective in overcoming chemoresistance towards carboplatin/paclitaxel. Conclusions: PDOs can be established from resectable NSCLC patients with high success rates, while preserving histopathological, cellular, and molecular characteristics of the matched primary tumors. Considering the time frames of growth and drug testing, PDOs can serve as clinically applicable drug response testing platforms to traditional and repurposed drugs. Citation Format: Yariswamy Manjunath, Suvilesh Kanve Nagaraj, Yulia I. Nussbaum, Mohamed Gadelkarim, Kevin F. Staveley-O’Carroll, Eric T. Kimchi, Guangfu Li, Wesley Warren, Chi-Ren Shyu, Matthew Ciorba, Jonathan B. Mitchem, Jussuf T. Kaifi. Patient-derived organoids from surgically treated, localized non-small cell lung cancer as high-throughput drug testing platforms for conventional and repurposed drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 205.

Colorectal cancer (CRC) remains one of the deadliest malignancies worldwide despite recent progress in screening and treatment strategies. Osteopontin (OPN) is a multifunctional protein expressed by multiple cells in the tumor microenvironment (TME) and is associated with immune suppression and poor prognosis in CRC. Tumor-associated macrophages (TAM), are also associated with immune suppression and poor prognosis in CRC, and may express OPN. While both TAM and OPN overlap in the TME, the role of OPN and OPN expressing TAM is poorly understood. In this study, we show the critical interaction and strategy for targeting this axis to overcome therapeutic resistance in CRC. Five publicly available CRC scRNA-seq data (571,818 cells total) from both untreated and treated patients were analyzed to determine the OPN expression and OPN-CD44 interaction in primary CRC and liver metastases using CellChat ligand-receptor interaction analysis. For experimental analyses, patient tissue was collected from the University of Missouri (MU) and the Harry S. Truman VA (HSTVA) on an approved IRB. OPN-positive TAMs were determined by immunofluorescence (IFC) and flow cytometry (FC). Analysis of FC data was completed using analyzed (FlowJo). Wilcoxon rank sum test was performed to find differentially expressed genes between the samples with and without OPN-CD44 interaction in each dataset. Data from scRNA-seq, IFC, and FC all demonstrated that TAMs were the predominant cells expressing OPN in the TME and the majority of TAM are OPN+. Most interestingly, in scRNA-seq data, we found minimal OPN+TAM in primary tumors from treated patients. These patients had a good response to therapy. Conversely, in FC data from our own patients, we found OPN+ TAM were preserved in primary tumors from patients’ post-therapy that had no response. Gene set enrichment analysis (GSEA) in treated patients with minimal OPN+TAM showed decreased exhaustion markers (logFC>0.5, FDR adjusted p-value<0.05). We then found that the strongest OPN-CD44 interactions were predicted between OPN+TAM and other TAM as well as T cells. Separating patients into high and low predicted OPN-CD44 interactors, GSEA demonstrated enrichment of cell death regulation and apoptosis pathways in CD8+ T cells in high interactors. To determine potential targets for modulation of OPN+TAM, we undertook trajectory analysis demonstrating that OPN+TAM were an intermediate phase, suggesting they are malleable. We then found that these OPN+TAM highly express TLR8 in both scRNAseq and FC, suggesting a potential target to modulate OPN+TAM. In this study, we demonstrate that OPN+TAM are the largest proportion of TAM in CRC tumors. Additionally, the presence of OPN+ TAM in treated samples is associated with response to systemic therapy. OPN+TAM appear to represent an intermediate phase of TAMs differentiation and highly express TLR8 suggesting a mechanism for modulation to improve patient response. Further ex vivo studies targeting these cells and their role in therapeutic response are critical. Citation Format: Yulia I Nussbaum, Yariswamy Manjunath, Elizabeth Shumway, Kaifi T Jussuf, Wesley C Warren, Jonathan B Mitchem. Studying the role of OPN-CD44 interaction in colorectal cancer antitumor immunity suppression using scRNA-seq data [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr B57.